Richard Lab:mRNA quantification

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Revision as of 13:57, 29 October 2011 by Michael A. Speer (talk | contribs) (RNA extraction)
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This page is for mRNA quantification;

RNA extraction

1.Use a kit or take a look at this hub page
2.Be really carefull when you do this, use DEPC water and some kind of RNAase degrading spray.

You can use a a reverse transcription PCR for this.

Reverse Transcription

1.Prepare the foloowing reaction mix (in this order):

  • 2μL Nuclease free water
  • 1μL Random hexamer mix (60μM)
  • 5μL AMV reaction mix
  • 1μL RNA from extraction (500-1000ng/μL)
  • 1μL AMV reverse transcriptase

2.Incubate at 25°C for 5 min. 3.Incubate at 42°C for 1 hour. 4.Inactivate enzyme at 80°C for 5 minutes. 5.Add 20μL nuclease free water. 6.Store at -20°C.